Directed evolution of thermophilic enzymes

ABSTRACT

Thermostable enzyme are subjected to mutagenesis to produce a thermophilic enzyme which is stable at thermophilic temperature and which has increased activities at least two-fold higher than the activity of the wild-type thermostable enzyme at lower temperatures, which are temperatures of 50° C. or lower.

The present invention relates to enzymes, particularly to thermostable enzymes. More particularly, the present invention relates to thermostable enzymes which are stable at high temperature and which have improved activity at lower temperatures.

Thermostable enzymes are enzymes that function at greater than 60° C. Thermostable enzymes are utilized in both industry and biomedical research in assays where certain steps of the assay are performed at significantly increased temperatures. Thermostable enzymes may be obtained from thermophilic organisms found in hot springs, volcanic origin, tropical areas etc. Examples of such organisms, for instance, include prokaryotic microorganisms, such as eubacteria and archaebacteria (Bronneomerier, K. and Staudenbauer, W. L., D. R. Woods (ed), the Clostridia and Biotechnology, Butterworth Publishers, Stoneham, M. A. (1993), among other organisms.

Thermostable enzymes exhibit greater storage life capacity and organic solvent resistance, as compared to their mesophilic counterparts.

There are applications in industry and in research for thermostable enzymes which exhibit enzyme activity at a desired minimum temperature. An example of this occurs in molecular diagnostics wherein reporter molecules must survive long term storage at room temperature or higher or they need to function in unusual environments, and the assays which employ them are performed at room temperature where the activity of thermostable enzymes is generally very low.

FIG. 1 illustrates the full length DNA sequence (SEQ ID NO:1) and corresponding deduced amino acid sequence (SEQ ID NO:2) Thermococcus 9N2 Beta-glycosidase.

Applicant has found that it is possible to provide thermostable enzymes which have improved activity at lower temperatures.

More particularly, Applicant has found that the activity of thermophilic enzymes can be improved at lower temperatures while maintaining the temperature stability of such enzymes.

Still more particularly, Applicant has found there can be obtained a thermostable enzyme with improved activity at lower temperature by subjecting to mutagenesis a thermostable enzyme or polynucleotide encoding such thermostable enzyme followed by a screening of the resulting mutants to identify a mutated enzyme or a mutated polynucleotide encoding a mutated enzyme, which mutated enzyme retains thermostability and which has an enzyme activity at lower temperatures which is at least two (2) times greater than a corresponding non-mutated enzyme.

The thermostable enzymes and mutated thermostable enzymes are stable at temperatures up to 60° C. and preferably are stable at temperatures of up to 70° C. and more preferably at temperatures up to 95° C. and higher.

Increased activity of mutated thermostable enzymes at lower temperatures is meant to encompass activities which are at least two-fold, preferably at least four-fold, and more preferably at least ten-fold greater than that of the corresponding wild-type enzyme.

Increased enzyme activity at lower temperatures means that enzyme activity is increased at a temperature below 50° C., preferably below 40° C. and more preferably below 10° C. Thus, in comparing enzyme activity at a lower temperature between the mutated and non-mutated enzyme, the enzyme activity of the mutated enzyme at defined lower temperatures is at least 2 times greater than the enzyme activity of the corresponding non-mutated enzyme.

Thus, lower temperatures and lower temperature ranges include temperatures which are at least 5° C. less than the temperature at which thermostable enzymes are stable, which includes temperatures below 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., 25° C. and 20° C. with below 50° C., being preferred, and below 40° C. being more preferred, and below 30° C. (or approximately room temperature) being most preferred.

In accordance with an aspect of the present invention, the lower temperature or lower temperature range at which a greater enzyme activity is desired is determined and a thermostable enzyme(s), or polynucleotide encoding such enzyme(s), are subjected to mutagenesis and the resulting mutants are screened to determine mutated enzymes (or polynucleotide encoding mutated enzymes) which retain thermostability and which have a minimum desired increase in enzyme activity at the desired temperature or temperature range.

Thermostable enzymes are enzymes which have activity, i.e. are not degraded, at temperatures above 60° C. Thermostable enzymes also have increased storage life, and high resistance to organic solvents.

Thermostable enzymes may be isolated from thermophilic organisms such as those which are found in elevated temperatures such as in hot springs, volcanic areas and tropical areas. Examples of thermophilic organisms are prokaryotic organisms for example, thermophilic bacteria such as eubacteria and archaebacteria.

The DNA from these thermostable organisms can then be isolated by available techniques that are described in the literature. The IsoQuick® nucleic acid extraction kit (MicroProbe Corporation) is suitable for this purpose.

The term "derived" or "isolated" means that material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated.

The DNA isolated or derived from these microorganisms can preferably be inserted into a vector. Such vectors are preferably those containing expression regulatory sequences, including promoters, enhancers and the like. Such polynucleotides can be part of a vector and/or a composition and still be isolated, in that such vector or composition is not part of its natural environment.

Alternatively, enzymes not known to have thermostable properties can be screened for such properties by inserting the DNA encoding the enzyme in an expression vector and transforming a suitable host as hereinafter described, such that the enzyme may be expressed and screened for positive thermostable activity.

As representative examples of expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, phosmids, bacterial artificial chromosomes, viral DNA (e.g. vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus, yeast, etc.) Thus, for example, the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9Qiagen), psiX174, pBluescript SK, pBluescript KS, (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pWLNEO, pXT1, pSG (Stratagene) pSVK3, pBPV, PMSG, pSVLSV40(Pharmacia). Any other plasmid or vector may be used as long as they are replicable and viable in the host.

The DNA derived from a microorganism(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operon encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the peroplasmic space or extracellular medium.

The DNA selected and isolated as hereinabove described is introduced into a suitable host to prepare a library which is screened for the desired enzyme activity. The selected DNA is preferably already in a vector which includes appropriate control sequences whereby selected DNA which encodes for an enzyme may be expressed, for detection of the desired activity. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by transformation, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

With particular references to various mammalian cell culture systems that can be employed to express recombinant protein, examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa 293 and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The isolated DNA encoding a thermostable enzyme is subjected to mutagenesis techniques, with the preferred type of mutagenesis techniques being set forth below.

The term "error-prone PCR" refers to a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Leung, D. W., et al., Technique, 1:11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33 (1922).

The term "oligonucleotide directed mutagenesis" refers to a process which allows for the generation of site-specific mutations in any cloned DNA segment of interest. Reidhaar-Olson, J. F. & Sauer, R. T., et al., Science, 241:53-57 (1988).

The term "assembly PCR" refers to a process which involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction.

The term "sexual PCR mutagenesis" refers to forced homologous recombination between DNA molecules of different but highly related DNA sequence in vitro, caused by random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction. Stemmer, W. P., PNAS, USA, 91:10747-10751 (1994)

The term "in vivo mutagenesis" refers to a process of generating random mutations in any cloned DNA of interest which involves the propagation of the DNA in a strain of E. coli that carries mutations in one or more of the DNA repair pathways. These "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA.

The term "cassette mutagenesis" refers to any process for replacing a small region of a double stranded DNA molecule with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains completely and/or partially randomized native sequence.

The term "recursive ensemble mutagenesis" refers to an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Arkin, A. P. and Youvan, D. C., PNAS, U.S.A., 89:7811-7815 (1992).

The term "exponential ensemble mutagenesis" refers to a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins, Delegrave, S. and Youvan, D. C Biotechnology Research, 11:1548-1552 (1993); and random and site-directed mutagenesis, Arnold, F. H., Current Opinion in Biotechnology, 4:450-455 (1993). All of the references mentioned above are hereby incorporated by reference in their entirety.

As can be seen from the above mutagenesis techniques, the DNA encoding an enzyme having the desired activity may be subject to mutagenesis alone, i.e. as naked DNA, or the DNA may be subjected to mutagenesis after insertion into an appropriate vector as hereinabove described. These techniques are referred to as in vitro mutagenesis.

Alternatively, in vivo mutagenesis may be performed wherein the DNA is subjected to mutagenesis while it is within a cell or living organism. A preferred example of this technique utilizes the XL1 Red Strain of E. coli (Stratagene, Inc.) which has its DNA repair genes, MutH, MutL and MutS, deleted such that many different mutations occur in a short time. Up to 10,000 mutations may take place within a 30 hour time span such that an entire mutated DNA library may be prepared from mutated DNA by procedures known in the art.

After an appropriate amount of time to allow mutations to take place, the mutated DNA is excised from the host cell in the case of in vivo mutagenesis and inserted in another appropriate vector and used to transform a non-mutator host, for example, XL1 Blue strain of E. coli after which a mutated DNA library is prepared. In the case of in vitro mutagenesis, if the mutated DNA has previously been inserted in an appropriate expression vector, said vector is then used directly to transform an appropriate non-mutator host for the preparation of a mutated DNA library, if the mutagenized DNA is not in an appropriate expression vector.

A library is prepared for screening by transforming a suitable organism. Hosts, particularly those specifically identified herein as preferred, are transformed by artificial introduction of the vectors containing the mutated DNA by inoculation under conditions conducive for such transformation.

The resultant libraries of transformed clones are then screened for clones which display activity for the enzyme of interest in a phenotypic assay for enzyme activity.

For example, having prepared a multiplicity of clones from DNA mutagenized by one of the techniques described above, such clones are screened for the specific enzyme activity of interest.

For example, the clones containing the mutated DNA are now subject to screening procedures to determine their activity within both higher temperatures and within the desired lower temperature range to identify mutants which have the desired increase in activity within the lower temperature range when compared to the corresponding wild-type thermostable enzyme which is non-mutated.

Positively identified clones, i.e. those which contain mutated DNA sequences which express thermostable enzymes which are thermostable and yet have an increased activity at least two times than the corresponding wild-type enzyme at temperatures within the lower temperature range, are isolated and sequenced to identify the DNA sequence. As an example, phosphatase activity at the desired lower temperature ranges may be identified by exposing the clones, and thus the thermostable enzyme and testing its ability to cleave an appropriate substrate.

In Example 1 phosphatase and β-galactosidase activity are measured by comparing the wild-type enzymes to the enzymes subjected to mutagenesis. As can be seen from the results of Example 1, mutagenesis of a wild-type phosphatase and β-galactosidase thermophilic enzyme produce mutated enzymes which were 3 and 2.5 times more active, respectively, at lower temperatures than the corresponding wild-type enzymes within the lower temperature range of room temperature.

In the case of protein engineering, after subjecting a thermophilic enzyme to mutagenesis, the mutagenized enzyme is screened for the desired activity namely, increased activity at lower temperatures while maintaining activity at the higher temperatures. Any of the known techniques for protein mutagenesis may be employed, with particularly preferred mutagenesis techniques being those discussed above.

As a representative list of enzymes which may be mutagenized in accordance with the aspects of the present invention, there may be mentioned, the following enzymes and their functions:

1 Lipase/Esterase

a. Enantioselective hydrolysis of esters (lipids)/thioesters

1) Resolution of racemic mixtures

2) Synthesis of optically active acids or alcohols from meso-diesters

b. Selective syntheses

1) Regiospecific hydrolysis of carbohydrate esters

2) Selective hydrolysis of cyclic secondary alcohols

a. Synthesis of optically active esters, lactones, acids, alcohols

1) Transesterification of activated/nonactivated esters

2) Interesterification

3) Optically active lactones from hydroxyesters

4) Regio- and enantioselective ring opening of anhydrides

d. Detergents

e. Fat/Oil conversion

f. Cheese ripening

2 Protease

a. Ester/amide synthesis

b. Peptide synthesis

c. Resolution of racemic mixtures of amino acid esters

d. Synthesis of non-natural amino acids

e. Detergents/protein hydrolysis

3 Glycosidase/Glycosyl transferase

a. Sugar/polymer synthesis

b. Cleavage of glycosidic linkages to form mono, di- and oligosaccharides

c. Synthesis of complex oligosaccharides

d. Glycoside synthesis using UDP-galactosyl transferase

e. Transglycosylation of disaccharides, glycosyl fluorides, aryl galactosides

f. Glycosyl transfer in oligosaccharide synthesis

g. Diastereoselective cleavage of β-glucosylsulfoxides

h. Asymmetric glycosylations

i. Food processing

j. Paper processing

4 Phosphatase/Kinase

a. Synthesis/hydrolysis of phosphate esters

1) Regio-, enantioselective phosphorylation

2) Introduction of phosphate esters

3) Synthesize phospholipid precursors

4) Controlled polynucleotide synthesis

b. Activate biological molecule

c. Selective phosphate bond formation without protecting groups

5 Mono/Dioxygenase

a. Direct oxyfunctionalization of unactivated organic substrates

b. Hydroxylation of alkane, aromatics, steroids

c. Epoxidation of alkenes

d. Enantioselective sulphoxidation

e. Regio- and stereoselective Bayer-Villiger oxidations

6 Haloperoxidase

a. Oxidative addition of halide ion to nucleophilic sites

b. Addition of hypohalous acids to olefinic bonds

c. Ring cleavage of cyclopropanes

d. Activated aromatic substrates converted to ortho and para derivatives

e. 1.3 diketones converted to 2-halo-derivatives

f. Heteroatom oxidation of sulfur and nitrogen containing substrates

g. Oxidation of enol acetates, alkynes and activated aromatic rings

7 Lignin peroxidase/Diarylpropane peroxidase

a. Oxidative cleavage of C--C bonds

b. Oxidation of benzylic alcohols to aldehydes

c. Hydroxylation of benzylic carbons

d. Phenol dimerization

e. Hydroxylation of double bonds to form diols

f. Cleavage of lignin aldehydes

8 Epoxide hydrolase

a. Synthesis of enantiomerically pure bioactive compounds

b. Regio- and enantioselective hydrolysis of epoxide

c. Aromatic and olefinic epoxidation by monooxygenases to form epoxides

d. Resolution of racemic epoxides

e. Hydrolysis of steroid epoxides

9 Nitrile hydratase/nitrilase

a. Hydrolysis of aliphatic nitriles to carboxamides

b. Hydrolysis of aromatic, heterocyclic, unsaturated aliphatic nitriles to corresponding acids

c. Hydrolysis of acrylonitrile

d. Production of aromatic and carboxamides, carboxylic acids (nicotinamide, picolinamide, isonicotinamide)

e. Regioselective hydrolysis of acrylic dinitrile

f. α-amino acids from α-hydroxynitriles

10 Transaminase

a. Transfer of amino groups into oxo-acids

11 Amidase/Acylase

a. Hydrolysis of amides, amidines, and other C--N bonds

b. Non-natural amino acid resolution and synthesis

The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplification's, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

EXAMPLE 1 Mutagenesis of Positive Enzyme Activity Clones

Mutagenesis was performed on two different enzymes (alkaline phosphatase and β-glycosidase), using the two different strategies described here, to generate new enzymes which exhibit a higher degree of activity at lower temperatures than the wild-type enzymes.

Alkaline Phosphatase

The XL1-Red strain (Stratagene) was transformed with DNA encoding an alkaline phosphatase (in plasmid pBluescript) from the organism OC9a according to the manufacturer's protocol. A 5 ml culture of LB+0.1 mg/ml ampicillin was inoculated with 200 μl of the transformation. The culture was allowed to grow at 37° C. for 30 hours. A miniprep was then performed on the culture, and screening was performed by transforming 2 μl of the resulting DNA into XL-1 Blue cells (Stratagene) according to the manufacturer's protocol.

Standard Alkaline Phosphatase Screening Assay

→Plate on LB/amp plates→Lift colonies with Duralon UV (Stratagene) or HATF (Millipore) membranes→Lyse in chloroform vapors for 30 seconds→Heat kill for 30 minutes at 85° C.→Develop filter at room temperature in BCIP buffer→Watch as filter develops and identify and pick fastest developing colonies ("positives")→Restreak "positives" onto a BCIP plate.

BCIP Buffer

20 mm CAPS pH 9.0

1 mm MgCl₂

0.01 mm ZnCl₂

0.1 mg/ml BCIP

The mutated OC9a phosphatase took 10 minutes to develop color and the wild type enzyme took 30 minutes to develop color in the screening assay.

Beta-Glycosidase

This protocol was used to mutagenize DNA encoding Thermococcus 9N2 Beta-Glycosidase. This DNA sequence is set forth in FIG. 1.

PCR

2 microliters dNTP's (10 mM Stocks)

10 microliters 10×PCR Buffer

0.5 microliters pBluescript vector containing Beta-glycosidase DNA (100 nanograms)

20 microliters 3' Primer (100 pmol)

20 microliters 5' Primer (100 pmol)

16 microliters MnCl 4H₂ O (1.25 mM Stock)

24.5 microliters H₂ O

1 microliter Taq Polymerase (5.0 Units)

100 microliters total

Reaction Cycle

95° C. 15 seconds

58° C. 30 seconds

72° C. 90 seconds

25 cycles (10 minute extension at 72° C.-4° C. incubation)

Run 5 microliters on a 1% agarose gel to check the reaction.

Purify on a Qiaquick column (Qiagen).

Resuspend in 50 microliters H₂ O.

Restriction Digest

25 microliters purified PCR product

10 microliters NEB Buffer #2

3 microliters Kpn I (10 U/microliter)

3 microliters EcoR1 (20 U/microliter)

59 microliters H₂ O

Cut for 2 hours at 37° C.

Purify on a Qiaquick column (Qiagen).

Elute with 35 microliters H₂ O.

Ligation

10 microliters Digested PCR product

5 microliters pBluescript Vector (cut with EcoRI/KpnI and phosphatased with shrimp alkaline phosphatase)

4 microliters 5× Ligation Buffer

1 microliter T4 DNA Ligase (BRL)

Ligate overnight.

Transform into M15pREP4 cells using electroporation.

Plate 100 or 200 microliters onto LB amp meth kan plates, grow overnight at 37 degrees celsius.

Beta-Glycosidase Assay

Perform glycosidase assay to screen for mutants as follows. The filter assay uses buffer Z (see recipe below) containing 1 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl-β-o-glucopyranoside (XGLU) (Diagnostic Chemicals Limited or Sigma).

Z-Buffer: (referenced in Miller, J. H. (1992) A Short Course in Bacterial Genetics, p. 445.)

per liter:

Na₂ HPO₄ -7H₂ O 16.1 g

NaH₂ PO₄ -H₂ O 5.5 g

KCl 0.75 g

MgSO₄ -7₂ O 0.246 g

β-mercaptoethanol 2.7 ml

(1) Adjust pH to 7.0

Perform colony lifts using Millipore HATF membrane filters.

(2) Lyse colonies with chloroform vapor in 150 mm glass petri dishes.

(3) Transfer filters to 100 mm glass petri dishes containing a piece of Whatman 3 MM filter paper saturated with Z buffer containing 1 mg/ml XGLU. After transferring filter bearing lysed colonies to the glass petri dish, maintain dish at room temperature.

(4) "Positives" were observed as blue spots on the filter membranes ("positives" are spots which appear early). Use the following filter rescue technique to retrieve plasmid from lysed positive colony. Use pasteur pipette (or glass capillary tube) to core blue spots on the filter membrane. Place the small filter disk in an Epp tube containing 20 μl water. Incubate the Epp tube at 75° C. for 5 minutes followed by vortexing to elute plasmid DNA off filter. Transform this DNA into electrocompetent E. coli cells. Repeat filter-lift assay on transformation plates to identify "positives." Return transformation plates to 37° C. incubator after filter lift to regenerate colonies. Inoculate 3 ml LBamp liquid with repurified positives and incubate at 37° C. overnight. Isolate plasmid DNA from these cultures and sequence plasmid insert.

The β-glycosidase subjected to mutagenesis acted on XGLU 2.5 times more efficiently than wild-type β-glycosidase.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 4                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4463 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Genomic DNA                                                (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Sequence                                                  (B) LOCATION: 1...4461                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AATTCCAGGATGAACCTCATCTGGTCGGTCTTGAGCTTGTACATTCCG48                             AsnSerArgMetAsnLeuIleTrpSerValLeuSerLeuTyrIlePro                               151015                                                                         GAACCGATGGGGTTCTCGCTGTTGGCGTACTTTATCGGGTCTTTGATG96                             GluProMetGlyPheSerLeuLeuAlaTyrPheIleGlySerLeuMet                               202530                                                                         TCCTTCCAGATGTGCTCAGGGACGATCGGGATCTGGAGCCAGTCCCAC144                            SerPheGlnMetCysSerGlyThrIleGlyIleTrpSerGlnSerHis                               354045                                                                         TCCGCGTGCGGATCGCTGAAGATGAAATCAACGGTTCTGTCGTCAACG192                            SerAlaCysGlySerLeuLysMetLysSerThrValLeuSerSerThr                               505560                                                                         ACCTTGACCTCCTTGAGCCAGCCCCAAACGCTCGAGAAGGAGGCACCG240                            ThrLeuThrSerLeuSerGlnProGlnThrLeuGluLysGluAlaPro                               65707580                                                                       GTGTGGTTCTTCGCGAGGAAGGTGAACTTAACGTCTTCAGCGGTTAGG288                            ValTrpPhePheAlaArgLysValAsnLeuThrSerSerAlaValArg                               859095                                                                         GGCTTTCCGTCCTGCCAGGTTAGGCCCTCCTTCAGCTTGACCTCGGTG336                            GlyPheProSerCysGlnValArgProSerPheSerLeuThrSerVal                               100105110                                                                      TTGTCGTTGATCCACTTCCCAGACTCGGCCAGCCAGGGAATGAGCTGG384                            LeuSerLeuIleHisPheProAspSerAlaSerGlnGlyMetSerTrp                               115120125                                                                      TCCTTCAGCGGGTCGAAGAACAGGGGCTCGATGAGGCTATCGTGCCTG432                            SerPheSerGlySerLysAsnArgGlySerMetArgLeuSerCysLeu                               130135140                                                                      CTGCGGCCCAGGAGACAAGGGGATAGTTCGTCGGCTGACTCCACAGAC480                            LeuArgProArgArgGlnGlyAspSerSerSerAlaAspSerThrAsp                               145150155160                                                                   CGCCTCCAACGTAGAGGGTTTCATTCCTGGGAAGCTCCTCGGCGCGGA528                            ArgLeuGlnArgArgGlyPheHisSerTrpGluAlaProArgArgGly                               165170175                                                                      CGTAGCCCGCGAATCCAACAAGGCTTGAAACCATCAGCACTGCCAGCA576                            ArgSerProArgIleGlnGlnGlyLeuLysProSerAlaLeuProAla                               180185190                                                                      GCAAACCAAGGATTCGTCTCATGCGCACCACCCCAGACCGCGAGGGTC624                            AlaAsnGlnGlyPheValSerCysAlaProProGlnThrAlaArgVal                               195200205                                                                      TGTAGTTATAAAAACGCGCTCCAAATTTATAAAACTTTGGGTTCTGTT672                            CysSerTyrLysAsnAlaLeuGlnIleTyrLysThrLeuGlySerVal                               210215220                                                                      ATAAAATTGGGGCAAAAATTAAAATCGGCAAACCTTATAAGGGAGAAA720                            IleLysLeuGlyGlnLysLeuLysSerAlaAsnLeuIleArgGluLys                               225230235240                                                                   GGCAAAGTTACATGGGGGTTGGTCTATGCTACCAGAAGGCTTTCTCTG768                            GlyLysValThrTrpGlyLeuValTyrAlaThrArgArgLeuSerLeu                               245250255                                                                      GGGCGTGTCCCAGTCCGGCTTTCAGTTCGAGATGGGCGACAAGCTCAG816                            GlyArgValProValArgLeuSerValArgAspGlyArgGlnAlaGln                               260265270                                                                      GAGGAACATTCCGAACACAGACTGGTGGAAGTGGGTCAGGGATCCCTT864                            GluGluHisSerGluHisArgLeuValGluValGlyGlnGlySerLeu                               275280285                                                                      CAACATAAAGAGGGAACTCGTCAGCGGCGACCTGCCCGAGGAGGGGAT912                            GlnHisLysGluGlyThrArgGlnArgArgProAlaArgGlyGlyAsp                               290295300                                                                      AAACAACTACGAACTTTACGAGAAGGATCACCGCCTCGCCAGAGACCT960                            LysGlnLeuArgThrLeuArgGluGlySerProProArgGlnArgPro                               305310315320                                                                   CGGTCTGAACGTTTACAGGATTGGAATAGAGTGGAGCAGGATCTTTCC1008                           ArgSerGluArgLeuGlnAspTrpAsnArgValGluGlnAspLeuSer                               325330335                                                                      CTGGCCAACGTGGTTTGTGGAGGTCGTGCGGGACAGCTACGGACTCGT1056                           LeuAlaAsnValValCysGlyGlyArgAlaGlyGlnLeuArgThrArg                               340345350                                                                      GAAGGACGTCAAAATCGAAGACACGCTCGAAGAGCTCGACGAGATAGC1104                           GluGlyArgGlnAsnArgArgHisAlaArgArgAlaArgArgAspSer                               355360365                                                                      GAATCATCAGGAGATAGCCTACTACCGCCGCGTTATAGAGCACCTCAG1152                           GluSerSerGlyAspSerLeuLeuProProArgTyrArgAlaProGln                               370375380                                                                      GGAGCTGGGCTTCAAGGTCATCGTGAACCTCAACCACTTCACGCTCCC1200                           GlyAlaGlyLeuGlnGlyHisArgGluProGlnProLeuHisAlaPro                               385390395400                                                                   CCTCTGGCTTCACGATCCGATAATCGCGAGGGAGAAGGCCCTCACCAA1248                           ProLeuAlaSerArgSerAspAsnArgGluGlyGluGlyProHisGln                               405410415                                                                      CGGGATTGGCTGGGTCGGGCAGGAGAGCGTGGTGGAGTTCGCCAAGTA1296                           ArgAspTrpLeuGlyArgAlaGlyGluArgGlyGlyValArgGlnVal                               420425430                                                                      CGCGGCGTACATCGCGAACGCACTCGGGGACCTCGTTATGTGGAGCAC1344                           ArgGlyValHisArgGluArgThrArgGlyProArgTyrValGluHis                               435440445                                                                      CTTCAACGAGCCGATGGTCGTTGTGGAGCTCGGTTACCTCGCGCCCTA1392                           LeuGlnArgAlaAspGlyArgCysGlyAlaArgLeuProArgAlaLeu                               450455460                                                                      CTCCGGCTTTCCGCCGGGGGTTATGAACCCCGAGGCGGCAAAGCTGGC1440                           LeuArgLeuSerAlaGlyGlyTyrGluProArgGlyGlyLysAlaGly                               465470475480                                                                   AATCCTCAACATGATAAACGCCCACGCACTGGCCTACAAGATGATAAA1488                           AsnProGlnHisAspLysArgProArgThrGlyLeuGlnAspAspLys                               485490495                                                                      GAAGTTCGACAGGGTAAAGGCCGAGGATTCCCGCTCCGAGGCCGAGGT1536                           GluValArgGlnGlyLysGlyArgGlyPheProLeuArgGlyArgGly                               500505510                                                                      CGGGATAATCTACAACAACATAGGCGTTGCCTATCCATACGACTCCAA1584                           ArgAspAsnLeuGlnGlnHisArgArgCysLeuSerIleArgLeuGln                               515520525                                                                      CGACCCAAAGGACGTGAAAGCTGCAGAAAACGACAACTACTTCCACAG1632                           ArgProLysGlyArgGluSerCysArgLysArgGlnLeuLeuProGln                               530535540                                                                      CGGGCTCTTCTTCGACGCAATCCACAAGGGCAAGCTCAACATCGAGTT1680                           ArgAlaLeuLeuArgArgAsnProGlnGlyGlnAlaGlnHisArgVal                               545550555560                                                                   CGACGGGACCTTCGTCAAAGTTCGGCATCTCAGGGGGAACGACTGGAT1728                           ArgArgAspLeuArgGlnSerSerAlaSerGlnGlyGluArgLeuAsp                               565570575                                                                      AGGCGTCTACTACACGAGAGAAGTCGTCAGGTATTCGGAGCCCAAGTT1776                           ArgArgLeuLeuHisGluArgSerArgGlnValPheGlyAlaGlnVal                               580585590                                                                      CCCGAGCATACCCCTGATATCCTTCCGGGGAGTTCACAACTACGGCTA1824                           ProGluHisThrProAspIleLeuProGlySerSerGlnLeuArgLeu                               595600605                                                                      CGCCTGCAGGCCCGGGAGTTCTTCCGCCGACGGAAGGCCCGTAAGCGA1872                           ArgLeuGlnAlaArgGluPhePheArgArgArgLysAlaArgLysArg                               610615620                                                                      CATCGGCTGGGAGATCTATCCGGAGGGGATCTACGACTCGATAAGAGA1920                           HisArgLeuGlyAspLeuSerGlyGlyAspLeuArgLeuAspLysArg                               625630635640                                                                   GGCCAACAAATACGGGGTCCCGGTTTACGTCACCGAAAACGGAATAGC1968                           GlyGlnGlnIleArgGlyProGlyLeuArgHisArgLysArgAsnSer                               645650655                                                                      CGATTCAACCACCCTGCGGCCGTACTACCTCGCGAGCCATGTAGCGAA2016                           ArgPheAsnHisProAlaAlaValLeuProArgGluProCysSerGlu                               660665670                                                                      GATGGAGGCGTACGAGGCGGGTTACGACGTCAGGGGCTACCTCTACTG2064                           AspGlyGlyValArgGlyGlyLeuArgArgGlnGlyLeuProLeuLeu                               675680685                                                                      GGCGCTGACCGACAACTACGAGTGGGCCCTCGGTTTCAGGATGAGGTT2112                           GlyAlaAspArgGlnLeuArgValGlyProArgPheGlnAspGluVal                               690695700                                                                      CGGCCTCTAAGTGGATCTCATAACCAAGGAGAGAACACCGCGGGAGGA2160                           ArgProLeuSerGlySerHisAsnGlnGlyGluAsnThrAlaGlyGly                               705710715720                                                                   AAGCGTAAAGGTTTAGGGCATCGTGGAGAACAACGGAGTGAGCAAGGA2208                           LysArgLysGlyLeuGlyHisArgGlyGluGlnArgSerGluGlnGly                               725730735                                                                      AATCCGGGAGAAGTTCGGACTTGGGTGAAGGTAATGAAGACGATAGCC2256                           AsnProGlyGluValArgThrTrpValLysValMetLysThrIleAla                               740745750                                                                      GTCGATGAGGACACTTGGGAGGCAAGAAGCAGGTCAGGCTTGAGGCAG2304                           ValAspGluAspThrTrpGluAlaArgSerArgSerGlyLeuArgGln                               755760765                                                                      ATCGTACGACGAAGTCCTGAAAAAGCTCATACAGGCCTGGACAGGGTT2352                           IleValArgArgSerProGluLysAlaHisThrGlyLeuAspArgVal                               770775780                                                                      GACTCGACAAGGCCGAGAGCGGCAACGACGAGGAGGCCGAGCTCATGC2400                           AspSerThrArgProArgAlaAlaThrThrArgArgProSerSerCys                               785790795800                                                                   TCAACCTCAAGAACAAGAAGACGGGAGGACAGGGTAATGAAGAGACTC2448                           SerThrSerArgThrArgArgArgGluAspArgValMetLysArgLeu                               805810815                                                                      CCTGAGAGGGTCTCTTTCGATCCCGAGGCGTTCGTTGAGATAAACCGA2496                           ProGluArgValSerPheAspProGluAlaPheValGluIleAsnArg                               820825830                                                                      AAGAGAAACAGGGACTTTTTAGAGTTCCTCTTGGCGGAGTTCCAGGTG2544                           LysArgAsnArgAspPheLeuGluPheLeuLeuAlaGluPheGlnVal                               835840845                                                                      NCGGTTTCCTTCTTCACGGTTCATCCATACCTCCTCGGCAAGACCTAT2592                           XaaValSerPhePheThrValHisProTyrLeuLeuGlyLysThrTyr                               850855860                                                                      CTGGGCAGGGACCTGGAAAGCGAAGTTCGGGCCCTCAACGAAGCCTAC2640                           LeuGlyArgAspLeuGluSerGluValArgAlaLeuAsnGluAlaTyr                               865870875880                                                                   ACCATCGTGTATCCCACGAAAGAACTCCTCATGAGGGCCATAGAAATC2688                           ThrIleValTyrProThrLysGluLeuLeuMetArgAlaIleGluIle                               885890895                                                                      GAGGCGAGGCTGATAAAAAGGGGAATTTTTCTCTCTTTCGACGACATC2736                           GluAlaArgLeuIleLysArgGlyIlePheLeuSerPheAspAspIle                               900905910                                                                      GTCATTGGAGTAACTGCCATTGAAAACAACGCCCTTCTCGTGAGCTCT2784                           ValIleGlyValThrAlaIleGluAsnAsnAlaLeuLeuValSerSer                               915920925                                                                      GCCCCCTCACGCTACAGGCCCCTTGAGAAGTACGGGCTCAACGTTATG2832                           AlaProSerArgTyrArgProLeuGluLysTyrGlyLeuAsnValMet                               930935940                                                                      GGGCTCAAGCTCCTTCTTCGACGAACTCCGGAAGCTCGCCCGGAAGGA2880                           GlyLeuLysLeuLeuLeuArgArgThrProGluAlaArgProGluGly                               945950955960                                                                   AGCCGCCAGATGGGAGGTGCCCCCGGTGGGATCTTCTCCAGAACGAGA2928                           SerArgGlnMetGlyGlyAlaProGlyGlyIlePheSerArgThrArg                               965970975                                                                      ATGAACGCTATCCTCGGGAAGAGCCAGCGGAGGCTTCTCGCCCTAAAG2976                           MetAsnAlaIleLeuGlyLysSerGlnArgArgLeuLeuAlaLeuLys                               980985990                                                                      CCCCTCTCCTCGAACTCCCCCCTAAGCTCCTCCGGTGAGGGAAAGGCG3024                           ProLeuSerSerAsnSerProLeuSerSerSerGlyGluGlyLysAla                               99510001005                                                                    TCTATCGAGCTGACAAGGTGTTCGGCTTTCTCGTTGCCCGTCGTCAGC3072                           SerIleGluLeuThrArgCysSerAlaPheSerLeuProValValSer                               101010151020                                                                   TTTCCGATGAGGGGAACAACCGTTCTCGTGAGCCAGGCCATCTTTCCA3120                           PheProMetArgGlyThrThrValLeuValSerGlnAlaIlePhePro                               1025103010351040                                                               AGGAGCGAGGGGTTCTTTGAGAACTCGAGGATTACGAGCCTTCCTCCC3168                           ArgSerGluGlyPhePheGluAsnSerArgIleThrSerLeuProPro                               104510501055                                                                   GGCTTCAGAACACGGTGGAGCTCCTCTATAGCTTTTTCCCTATCGGAG3216                           GlyPheArgThrArgTrpSerSerSerIleAlaPheSerLeuSerGlu                               106010651070                                                                   AAGNTTCTGAGGTCGGAGGCGACGCTGACAATGTCGNAGCTCCCGTCC3264                           LysXaaLeuArgSerGluAlaThrLeuThrMetSerXaaLeuProSer                               107510801085                                                                   GGAGNCATTTCTTCCGCCCTGCCAACGCTCAGCCTCGCGNAGGGGACC3312                           GlyXaaIleSerSerAlaLeuProThrLeuSerLeuAlaXaaGlyThr                               109010951100                                                                   TTTCTCCCCGCTATCCTGAGCATCTCCTCGCTGCAGTCGAGGCCGAAC3360                           PheLeuProAlaIleLeuSerIleSerSerLeuGlnSerArgProAsn                               1105111011151120                                                               TACGCCCGACAGGTTTCTCTTTTCAAGCCTCTTCCTCATGCAGAGCAT3408                           TyrAlaArgGlnValSerLeuPheLysProLeuProHisAlaGluHis                               112511301135                                                                   CATGTCCCTGGTTCCGCAGGCCACGTCAAGGATTTTCGGCCTTTCGCG3456                           HisValProGlySerAlaGlyHisValLysAspPheArgProPheAla                               114011451150                                                                   AACCTCAAGGGACTTCAAAACCTCCTCGCAGGCCTTTTTCCTCCACAA3504                           AsnLeuLysGlyLeuGlnAsnLeuLeuAlaGlyLeuPheProProGln                               115511601165                                                                   CCTGTCGAGACTGAGGCTTATCAGCCTGTTGGTATCGTAGCGCTCCGC3552                           ProValGluThrGluAlaTyrGlnProValGlyIleValAlaLeuArg                               117011751180                                                                   AATGCTGTCAAAGAGCTCCCTTACCAAGCTCCTCCCTCCCGAGGACCT3600                           AsnAlaValLysGluLeuProTyrGlnAlaProProSerArgGlyPro                               1185119011951200                                                               TCTTTATCTTCGCGGGCCTTCCGCCTAGGTAAACCCTGTCCGCAATTT3648                           SerLeuSerSerArgAlaPheArgLeuGlyLysProCysProGlnPhe                               120512101215                                                                   AGAGCTCCTCGAGCTGGTGCGAGACGACCAGAACGCCTTTTCCAGAAT3696                           ArgAlaProArgAlaGlyAlaArgArgProGluArgLeuPheGlnAsn                               122012251230                                                                   TGGCTAATTCCCGAATTATGGAAAGAAGTTCTTCTTTGGAGCGCGCGT3744                           TrpLeuIleProGluLeuTrpLysGluValLeuLeuTrpSerAlaArg                               123512401245                                                                   CGAGGCCGGAGGGCTCGTCCAGAATCAGGACATCGAAGTCCAGCAGGG3792                           ArgGlyArgArgAlaArgProGluSerGlyHisArgSerProAlaGly                               125012551260                                                                   CGCGCAGGAGAGAAGTTTTTCTCCTCGTCCCCCCGCTCACTTCCTTTG3840                           ArgAlaGlyGluLysPhePheSerSerSerProArgSerLeuProLeu                               1265127012751280                                                               GATAGAGGTCGAGGTAGTTCTCCAGGCCGAGTCTCTCCACGTATTCGA3888                           AspArgGlyArgGlySerSerProGlyArgValSerProArgIleArg                               128512901295                                                                   GCCTGCACTCCCTTCCCCAGCGGGCCGGCAGGCAGACGTTGTCCCGCC3936                           AlaCysThrProPheProSerGlyProAlaGlyArgArgCysProAla                               130013051310                                                                   TCTTCCACGGCAGAAGGTAGTCCTCCTGATAGAGGACGGAGGGGTTCT3984                           SerSerThrAlaGluGlySerProProAspArgGlyArgArgGlySer                               131513201325                                                                   TCACAAAGACCTCGCCCGAGTCCGGGTTCTCAACTCCGGCCAAAATCT4032                           SerGlnArgProArgProSerProGlySerGlnLeuArgProLysSer                               133013351340                                                                   TCACGAGGGTGCTCTTTCCCGTCCCGTTCGGCCCGAGGCCGACCACCT4080                           SerArgGlyCysSerPheProSerArgSerAlaArgGlyArgProPro                               1345135013551360                                                               CGCCGGCCCCCTCAAGGCCCCCGTCGAGTATGGGCTCACAGGACTTTC4128                           ArgArgProProGlnGlyProArgArgValTrpAlaHisArgThrPhe                               136513701375                                                                   GGTTCTTCACCAGTACCAGCCTTTCACCCATTCCTCGACCTCCTCAGA4176                           GlySerSerProValProAlaPheHisProPheLeuAspLeuLeuArg                               138013851390                                                                   AGTAGCTGGTCGAGGGTTATCATGAGCAGGATTAAGAGCAGTGCCCAG4224                           SerSerTrpSerArgValIleMetSerArgIleLysSerSerAlaGln                               139514001405                                                                   GCAAAAACACCGGCTTTAATTCCCAAATCGGAGACGAGCTGGCCGATT4272                           AlaLysThrProAlaLeuIleProLysSerGluThrSerTrpProIle                               141014151420                                                                   CCTCCCGCTGAACCAAAAGCCTCGGCAACGACGCTTATCCTGAGCGCT4320                           ProProAlaGluProLysAlaSerAlaThrThrLeuIleLeuSerAla                               1425143014351440                                                               ATTCCCAGGGCGACTCTGCCTGCCGAGACCATCTCGGGGAGCGTTCCC4368                           IleProArgAlaThrLeuProAlaGluThrIleSerGlySerValPro                               144514501455                                                                   GGGACGATGAAGTGCCGGAGCAGCTTTGAGGGTTTGAGGAGAACTATC4416                           GlyThrMetLysCysArgSerSerPheGluGlyLeuArgArgThrIle                               146014651470                                                                   AGCGGGCGGTATTTTTCTATCACCTTTTCGCTTGAGCTCACTCCAGC4463                            SerGlyArgTyrPheSerIleThrPheSerLeuGluLeuThrPro                                  147514801485                                                                   (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1487 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (v) FRAGMENT TYPE: internal                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        AsnSerArgMetAsnLeuIleTrpSerValLeuSerLeuTyrIlePro                               151015                                                                         GluProMetGlyPheSerLeuLeuAlaTyrPheIleGlySerLeuMet                               202530                                                                         SerPheGlnMetCysSerGlyThrIleGlyIleTrpSerGlnSerHis                               354045                                                                         SerAlaCysGlySerLeuLysMetLysSerThrValLeuSerSerThr                               505560                                                                         ThrLeuThrSerLeuSerGlnProGlnThrLeuGluLysGluAlaPro                               65707580                                                                       ValTrpPhePheAlaArgLysValAsnLeuThrSerSerAlaValArg                               859095                                                                         GlyPheProSerCysGlnValArgProSerPheSerLeuThrSerVal                               100105110                                                                      LeuSerLeuIleHisPheProAspSerAlaSerGlnGlyMetSerTrp                               115120125                                                                      SerPheSerGlySerLysAsnArgGlySerMetArgLeuSerCysLeu                               130135140                                                                      LeuArgProArgArgGlnGlyAspSerSerSerAlaAspSerThrAsp                               145150155160                                                                   ArgLeuGlnArgArgGlyPheHisSerTrpGluAlaProArgArgGly                               165170175                                                                      ArgSerProArgIleGlnGlnGlyLeuLysProSerAlaLeuProAla                               180185190                                                                      AlaAsnGlnGlyPheValSerCysAlaProProGlnThrAlaArgVal                               195200205                                                                      CysSerTyrLysAsnAlaLeuGlnIleTyrLysThrLeuGlySerVal                               210215220                                                                      IleLysLeuGlyGlnLysLeuLysSerAlaAsnLeuIleArgGluLys                               225230235240                                                                   GlyLysValThrTrpGlyLeuValTyrAlaThrArgArgLeuSerLeu                               245250255                                                                      GlyArgValProValArgLeuSerValArgAspGlyArgGlnAlaGln                               260265270                                                                      GluGluHisSerGluHisArgLeuValGluValGlyGlnGlySerLeu                               275280285                                                                      GlnHisLysGluGlyThrArgGlnArgArgProAlaArgGlyGlyAsp                               290295300                                                                      LysGlnLeuArgThrLeuArgGluGlySerProProArgGlnArgPro                               305310315320                                                                   ArgSerGluArgLeuGlnAspTrpAsnArgValGluGlnAspLeuSer                               325330335                                                                      LeuAlaAsnValValCysGlyGlyArgAlaGlyGlnLeuArgThrArg                               340345350                                                                      GluGlyArgGlnAsnArgArgHisAlaArgArgAlaArgArgAspSer                               355360365                                                                      GluSerSerGlyAspSerLeuLeuProProArgTyrArgAlaProGln                               370375380                                                                      GlyAlaGlyLeuGlnGlyHisArgGluProGlnProLeuHisAlaPro                               385390395400                                                                   ProLeuAlaSerArgSerAspAsnArgGluGlyGluGlyProHisGln                               405410415                                                                      ArgAspTrpLeuGlyArgAlaGlyGluArgGlyGlyValArgGlnVal                               420425430                                                                      ArgGlyValHisArgGluArgThrArgGlyProArgTyrValGluHis                               435440445                                                                      LeuGlnArgAlaAspGlyArgCysGlyAlaArgLeuProArgAlaLeu                               450455460                                                                      LeuArgLeuSerAlaGlyGlyTyrGluProArgGlyGlyLysAlaGly                               465470475480                                                                   AsnProGlnHisAspLysArgProArgThrGlyLeuGlnAspAspLys                               485490495                                                                      GluValArgGlnGlyLysGlyArgGlyPheProLeuArgGlyArgGly                               500505510                                                                      ArgAspAsnLeuGlnGlnHisArgArgCysLeuSerIleArgLeuGln                               515520525                                                                      ArgProLysGlyArgGluSerCysArgLysArgGlnLeuLeuProGln                               530535540                                                                      ArgAlaLeuLeuArgArgAsnProGlnGlyGlnAlaGlnHisArgVal                               545550555560                                                                   ArgArgAspLeuArgGlnSerSerAlaSerGlnGlyGluArgLeuAsp                               565570575                                                                      ArgArgLeuLeuHisGluArgSerArgGlnValPheGlyAlaGlnVal                               580585590                                                                      ProGluHisThrProAspIleLeuProGlySerSerGlnLeuArgLeu                               595600605                                                                      ArgLeuGlnAlaArgGluPhePheArgArgArgLysAlaArgLysArg                               610615620                                                                      HisArgLeuGlyAspLeuSerGlyGlyAspLeuArgLeuAspLysArg                               625630635640                                                                   GlyGlnGlnIleArgGlyProGlyLeuArgHisArgLysArgAsnSer                               645650655                                                                      ArgPheAsnHisProAlaAlaValLeuProArgGluProCysSerGlu                               660665670                                                                      AspGlyGlyValArgGlyGlyLeuArgArgGlnGlyLeuProLeuLeu                               675680685                                                                      GlyAlaAspArgGlnLeuArgValGlyProArgPheGlnAspGluVal                               690695700                                                                      ArgProLeuSerGlySerHisAsnGlnGlyGluAsnThrAlaGlyGly                               705710715720                                                                   LysArgLysGlyLeuGlyHisArgGlyGluGlnArgSerGluGlnGly                               725730735                                                                      AsnProGlyGluValArgThrTrpValLysValMetLysThrIleAla                               740745750                                                                      ValAspGluAspThrTrpGluAlaArgSerArgSerGlyLeuArgGln                               755760765                                                                      IleValArgArgSerProGluLysAlaHisThrGlyLeuAspArgVal                               770775780                                                                      AspSerThrArgProArgAlaAlaThrThrArgArgProSerSerCys                               785790795800                                                                   SerThrSerArgThrArgArgArgGluAspArgValMetLysArgLeu                               805810815                                                                      ProGluArgValSerPheAspProGluAlaPheValGluIleAsnArg                               820825830                                                                      LysArgAsnArgAspPheLeuGluPheLeuLeuAlaGluPheGlnVal                               835840845                                                                      XaaValSerPhePheThrValHisProTyrLeuLeuGlyLysThrTyr                               850855860                                                                      LeuGlyArgAspLeuGluSerGluValArgAlaLeuAsnGluAlaTyr                               865870875880                                                                   ThrIleValTyrProThrLysGluLeuLeuMetArgAlaIleGluIle                               885890895                                                                      GluAlaArgLeuIleLysArgGlyIlePheLeuSerPheAspAspIle                               900905910                                                                      ValIleGlyValThrAlaIleGluAsnAsnAlaLeuLeuValSerSer                               915920925                                                                      AlaProSerArgTyrArgProLeuGluLysTyrGlyLeuAsnValMet                               930935940                                                                      GlyLeuLysLeuLeuLeuArgArgThrProGluAlaArgProGluGly                               945950955960                                                                   SerArgGlnMetGlyGlyAlaProGlyGlyIlePheSerArgThrArg                               965970975                                                                      MetAsnAlaIleLeuGlyLysSerGlnArgArgLeuLeuAlaLeuLys                               980985990                                                                      ProLeuSerSerAsnSerProLeuSerSerSerGlyGluGlyLysAla                               99510001005                                                                    SerIleGluLeuThrArgCysSerAlaPheSerLeuProValValSer                               101010151020                                                                   PheProMetArgGlyThrThrValLeuValSerGlnAlaIlePhePro                               1025103010351040                                                               ArgSerGluGlyPhePheGluAsnSerArgIleThrSerLeuProPro                               104510501055                                                                   GlyPheArgThrArgTrpSerSerSerIleAlaPheSerLeuSerGlu                               106010651070                                                                   LysXaaLeuArgSerGluAlaThrLeuThrMetSerXaaLeuProSer                               107510801085                                                                   GlyXaaIleSerSerAlaLeuProThrLeuSerLeuAlaXaaGlyThr                               109010951100                                                                   PheLeuProAlaIleLeuSerIleSerSerLeuGlnSerArgProAsn                               1105111011151120                                                               TyrAlaArgGlnValSerLeuPheLysProLeuProHisAlaGluHis                               112511301135                                                                   HisValProGlySerAlaGlyHisValLysAspPheArgProPheAla                               114011451150                                                                   AsnLeuLysGlyLeuGlnAsnLeuLeuAlaGlyLeuPheProProGln                               115511601165                                                                   ProValGluThrGluAlaTyrGlnProValGlyIleValAlaLeuArg                               117011751180                                                                   AsnAlaValLysGluLeuProTyrGlnAlaProProSerArgGlyPro                               1185119011951200                                                               SerLeuSerSerArgAlaPheArgLeuGlyLysProCysProGlnPhe                               120512101215                                                                   ArgAlaProArgAlaGlyAlaArgArgProGluArgLeuPheGlnAsn                               122012251230                                                                   TrpLeuIleProGluLeuTrpLysGluValLeuLeuTrpSerAlaArg                               123512401245                                                                   ArgGlyArgArgAlaArgProGluSerGlyHisArgSerProAlaGly                               125012551260                                                                   ArgAlaGlyGluLysPhePheSerSerSerProArgSerLeuProLeu                               1265127012751280                                                               AspArgGlyArgGlySerSerProGlyArgValSerProArgIleArg                               128512901295                                                                   AlaCysThrProPheProSerGlyProAlaGlyArgArgCysProAla                               130013051310                                                                   SerSerThrAlaGluGlySerProProAspArgGlyArgArgGlySer                               131513201325                                                                   SerGlnArgProArgProSerProGlySerGlnLeuArgProLysSer                               133013351340                                                                   SerArgGlyCysSerPheProSerArgSerAlaArgGlyArgProPro                               1345135013551360                                                               ArgArgProProGlnGlyProArgArgValTrpAlaHisArgThrPhe                               136513701375                                                                   GlySerSerProValProAlaPheHisProPheLeuAspLeuLeuArg                               138013851390                                                                   SerSerTrpSerArgValIleMetSerArgIleLysSerSerAlaGln                               139514001405                                                                   AlaLysThrProAlaLeuIleProLysSerGluThrSerTrpProIle                               141014151420                                                                   ProProAlaGluProLysAlaSerAlaThrThrLeuIleLeuSerAla                               1425143014351440                                                               IleProArgAlaThrLeuProAlaGluThrIleSerGlySerValPro                               144514501455                                                                   GlyThrMetLysCysArgSerSerPheGluGlyLeuArgArgThrIle                               146014651470                                                                   SerGlyArgTyrPheSerIleThrPheSerLeuGluLeuThrPro                                  147514801485                                                                   (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4463 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Genomic DNA                                                (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Sequence                                                  (B) LOCATION: 1...4461                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        AATTCCAGGATGAACCTCATCTGGTCGGTCTTGAGCTTGTACATTCCG48                             AsnSerArgMetAsnLeuIleTrpSerValLeuSerLeuTyrIlePro                               151015                                                                         GAACCGATGGGGTTCTCGCTGTTGGCGTACTTTATCGGGTCTTTGATG96                             GluProMetGlyPheSerLeuLeuAlaTyrPheIleGlySerLeuMet                               202530                                                                         TCCTTCCAGATGTGCTCAGGGACGATCGGGATCTGGAGCCAGTCCCAC144                            SerPheGlnMetCysSerGlyThrIleGlyIleTrpSerGlnSerHis                               354045                                                                         TCCGCGTGCGGATCGCTGAAGATGAAATCAACGGTTCTGTCGTCAACG192                            SerAlaCysGlySerLeuLysMetLysSerThrValLeuSerSerThr                               505560                                                                         ACCTTGACCTCCTTGAGCCAGCCCCAAACGCTCGAGAAGGAGGCACCG240                            ThrLeuThrSerLeuSerGlnProGlnThrLeuGluLysGluAlaPro                               65707580                                                                       GTGTGGTTCTTCGCGAGGAAGGTGAACTTAACGTCTTCAGCGGTTAGG288                            ValTrpPhePheAlaArgLysValAsnLeuThrSerSerAlaValArg                               859095                                                                         GGCTTTCCGTCCTGCCAGGTTAGGCCCTCCTTCAGCTTGACCTCGGTG336                            GlyPheProSerCysGlnValArgProSerPheSerLeuThrSerVal                               100105110                                                                      TTGTCGTTGATCCACTTCCCAGACTCGGCCAGCCAGGGAATGAGCTGG384                            LeuSerLeuIleHisPheProAspSerAlaSerGlnGlyMetSerTrp                               115120125                                                                      TCCTTCAGCGGGTCGAAGAACAGGGGCTCGATGAGGCTATCGTGCCTG432                            SerPheSerGlySerLysAsnArgGlySerMetArgLeuSerCysLeu                               130135140                                                                      CTGCGGCCCAGGAGACAAGGGGATAGTTCGTCGGCTGACTCCACAGAC480                            LeuArgProArgArgGlnGlyAspSerSerSerAlaAspSerThrAsp                               145150155160                                                                   CGCCTCCAACGTAGAGGGTTTCATTCCTGGGAAGCTCCTCGGCGCGGA528                            ArgLeuGlnArgArgGlyPheHisSerTrpGluAlaProArgArgGly                               165170175                                                                      CGTAGCCCGCGAATCCAACAAGGCTTGAAACCATCAGCACTGCCAGCA576                            ArgSerProArgIleGlnGlnGlyLeuLysProSerAlaLeuProAla                               180185190                                                                      GCAAACCAAGGATTCGTCTCATGCGCACCACCCCAGACCGCGAGGGTC624                            AlaAsnGlnGlyPheValSerCysAlaProProGlnThrAlaArgVal                               195200205                                                                      TGTAGTTATAAAAACGCGCTCCAAATTTATAAAACTTTGGGTTCTGTT672                            CysSerTyrLysAsnAlaLeuGlnIleTyrLysThrLeuGlySerVal                               210215220                                                                      ATAAAATTGGGGCAAAAATTAAAATCGGCAAACCTTATAAGGGAGAAA720                            IleLysLeuGlyGlnLysLeuLysSerAlaAsnLeuIleArgGluLys                               225230235240                                                                   GGCAAAGTTACATGGGGGTTGGTCTATGCTACCAGAAGGCTTTCTCTG768                            GlyLysValThrTrpGlyLeuValTyrAlaThrArgArgLeuSerLeu                               245250255                                                                      GGGCGTGTCCCAGTCCGGCTTTCAGTTCGAGATGGGCGACAAGCTCAG816                            GlyArgValProValArgLeuSerValArgAspGlyArgGlnAlaGln                               260265270                                                                      GAGGAACATTCCGAACACAGACTGGTGGAAGTGGGTCAGGGATCCCTT864                            GluGluHisSerGluHisArgLeuValGluValGlyGlnGlySerLeu                               275280285                                                                      CAACATAAAGAGGGAACTCGTCAGCGGCGACCTGCCCGAGGAGGGGAT912                            GlnHisLysGluGlyThrArgGlnArgArgProAlaArgGlyGlyAsp                               290295300                                                                      AAACAACTACGAACTTTACGAGAAGGATCACCGCCTCGCCAGAGACCT960                            LysGlnLeuArgThrLeuArgGluGlySerProProArgGlnArgPro                               305310315320                                                                   CGGTCTGAACGTTTACAGGATTGGAATAGAGTGGAGCAGGATCTTTCC1008                           ArgSerGluArgLeuGlnAspTrpAsnArgValGluGlnAspLeuSer                               325330335                                                                      CTGGCCAACGTGGTTTGTGGAGGTCGTGCGGGACAGCTACGGACTCGT1056                           LeuAlaAsnValValCysGlyGlyArgAlaGlyGlnLeuArgThrArg                               340345350                                                                      GAAGGACGTCAAAATCGAAGACACGCTCGAAGAGCTCGACGAGATAGC1104                           GluGlyArgGlnAsnArgArgHisAlaArgArgAlaArgArgAspSer                               355360365                                                                      GAATCATCAGGAGATAGCCTACTACCGCCGCGTTATAGAGCACCTCAG1152                           GluSerSerGlyAspSerLeuLeuProProArgTyrArgAlaProGln                               370375380                                                                      GGAGCTGGGCTTCAAGGTCATCGTGAACCTCAACCACTTCACGCTCCC1200                           GlyAlaGlyLeuGlnGlyHisArgGluProGlnProLeuHisAlaPro                               385390395400                                                                   CCTCTGGCTTCACGATCCGATAATCGCGAGGGAGAAGGCCCTCACCAA1248                           ProLeuAlaSerArgSerAspAsnArgGluGlyGluGlyProHisGln                               405410415                                                                      CGGGATTGGCTGGGTCGGGCAGGAGAGCGTGGTGGAGTTCGCCAAGTA1296                           ArgAspTrpLeuGlyArgAlaGlyGluArgGlyGlyValArgGlnVal                               420425430                                                                      CGCGGCGTACATCGCGAACGCACTCGGGGACCTCGTTATGTGGAGCAC1344                           ArgGlyValHisArgGluArgThrArgGlyProArgTyrValGluHis                               435440445                                                                      CTTCAACGAGCCGATGGTCGTTGTGGAGCTCGGTTACCTCGCGCCCTA1392                           LeuGlnArgAlaAspGlyArgCysGlyAlaArgLeuProArgAlaLeu                               450455460                                                                      CTCCGGCTTTCCGCCGGGGGTTATGAACCCCGAGGCGGCAAAGCTGGC1440                           LeuArgLeuSerAlaGlyGlyTyrGluProArgGlyGlyLysAlaGly                               465470475480                                                                   AATCCTCAACATGATAAACGCCCACGCACTGGCCTACAAGATGATAAA1488                           AsnProGlnHisAspLysArgProArgThrGlyLeuGlnAspAspLys                               485490495                                                                      GAAGTTCGACAGGGTAAAGGCCGAGGATTCCCGCTCCGAGGCCGAGGT1536                           GluValArgGlnGlyLysGlyArgGlyPheProLeuArgGlyArgGly                               500505510                                                                      CGGGATAATCTACAACAACATAGGCGTTGCCTATCCATACGACTCCAA1584                           ArgAspAsnLeuGlnGlnHisArgArgCysLeuSerIleArgLeuGln                               515520525                                                                      CGACCCAAAGGACGTGAAAGCTGCAGAAAACGACAACTACTTCCACAG1632                           ArgProLysGlyArgGluSerCysArgLysArgGlnLeuLeuProGln                               530535540                                                                      CGGGCTCTTCTTCGACGCAATCCACAAGGGCAAGCTCAACATCGAGTT1680                           ArgAlaLeuLeuArgArgAsnProGlnGlyGlnAlaGlnHisArgVal                               545550555560                                                                   CGACGGGACCTTCGTCAAAGTTCGGCATCTCAGGGGGAACGACTGGAT1728                           ArgArgAspLeuArgGlnSerSerAlaSerGlnGlyGluArgLeuAsp                               565570575                                                                      AGGCGTCTACTACACGAGAGAAGTCGTCAGGTATTCGGAGCCCAAGTT1776                           ArgArgLeuLeuHisGluArgSerArgGlnValPheGlyAlaGlnVal                               580585590                                                                      CCCGAGCATACCCCTGATATCCTTCCGGGGAGTTCACAACTACGGCTA1824                           ProGluHisThrProAspIleLeuProGlySerSerGlnLeuArgLeu                               595600605                                                                      CGCCTGCAGGCCCGGGAGTTCTTCCGCCGACGGAAGGCCCGTAAGCGA1872                           ArgLeuGlnAlaArgGluPhePheArgArgArgLysAlaArgLysArg                               610615620                                                                      CATCGGCTGGGAGATCTATCCGGAGGGGATCTACGACTCGATAAGAGA1920                           HisArgLeuGlyAspLeuSerGlyGlyAspLeuArgLeuAspLysArg                               625630635640                                                                   GGCCAACAAATACGGGGTCCCGGTTTACGTCACCGAAAACGGAATAGC1968                           GlyGlnGlnIleArgGlyProGlyLeuArgHisArgLysArgAsnSer                               645650655                                                                      CGATTCAACCACCCTGCGGCCGTACTACCTCGCGAGCCATGTAGCGAA2016                           ArgPheAsnHisProAlaAlaValLeuProArgGluProCysSerGlu                               660665670                                                                      GATGGAGGCGTACGAGGCGGGTTACGACGTCAGGGGCTACCTCTACTG2064                           AspGlyGlyValArgGlyGlyLeuArgArgGlnGlyLeuProLeuLeu                               675680685                                                                      GGCGCTGACCGACAACTACGAGTGGGCCCTCGGTTTCAGGATGAGGTT2112                           GlyAlaAspArgGlnLeuArgValGlyProArgPheGlnAspGluVal                               690695700                                                                      CGGCCTCTAAGTGGATCTCATAACCAAGGAGAGAACACCGCGGGAGGA2160                           ArgProLeuSerGlySerHisAsnGlnGlyGluAsnThrAlaGlyGly                               705710715720                                                                   AAGCGTAAAGGTTTAGGGCATCGTGGAGAACAACGGAGTGAGCAAGGA2208                           LysArgLysGlyLeuGlyHisArgGlyGluGlnArgSerGluGlnGly                               725730735                                                                      AATCCGGGAGAAGTTCGGACTTGGGTGAAGGTAATGAAGACGATAGCC2256                           AsnProGlyGluValArgThrTrpValLysValMetLysThrIleAla                               740745750                                                                      GTCGATGAGGACACTTGGGAGGCAAGAAGCAGGTCAGGCTTGAGGCAG2304                           ValAspGluAspThrTrpGluAlaArgSerArgSerGlyLeuArgGln                               755760765                                                                      ATCGTACGACGAAGTCCTGAAAAAGCTCATACAGGCCTGGACAGGGTT2352                           IleValArgArgSerProGluLysAlaHisThrGlyLeuAspArgVal                               770775780                                                                      GACTCGACAAGGCCGAGAGCGGCAACGACGAGGAGGCCGAGCTCATGC2400                           AspSerThrArgProArgAlaAlaThrThrArgArgProSerSerCys                               785790795800                                                                   TCAACCTCAAGAACAAGAAGACGGGAGGACAGGGTAATGAAGAGACTC2448                           SerThrSerArgThrArgArgArgGluAspArgValMetLysArgLeu                               805810815                                                                      CCTGAGAGGGTCTCTTTCGATCCCGAGGCGTTCGTTGAGATAAACCGA2496                           ProGluArgValSerPheAspProGluAlaPheValGluIleAsnArg                               820825830                                                                      AAGAGAAACAGGGACTTTTTAGAGTTCCTCTTGGCGGAGTTCCAGGTG2544                           LysArgAsnArgAspPheLeuGluPheLeuLeuAlaGluPheGlnVal                               835840845                                                                      NCGGTTTCCTTCTTCACGGTTCATCCATACCTCCTCGGCAAGACCTAT2592                           XaaValSerPhePheThrValHisProTyrLeuLeuGlyLysThrTyr                               850855860                                                                      CTGGGCAGGGACCTGGAAAGCGAAGTTCGGGCCCTCAACGAAGCCTAC2640                           LeuGlyArgAspLeuGluSerGluValArgAlaLeuAsnGluAlaTyr                               865870875880                                                                   ACCATCGTGTATCCCACGAAAGAACTCCTCATGAGGGCCATAGAAATC2688                           ThrIleValTyrProThrLysGluLeuLeuMetArgAlaIleGluIle                               885890895                                                                      GAGGCGAGGCTGATAAAAAGGGGAATTTTTCTCTCTTTCGACGACATC2736                           GluAlaArgLeuIleLysArgGlyIlePheLeuSerPheAspAspIle                               900905910                                                                      GTCATTGGAGTAACTGCCATTGAAAACAACGCCCTTCTCGTGAGCTCT2784                           ValIleGlyValThrAlaIleGluAsnAsnAlaLeuLeuValSerSer                               915920925                                                                      GCCCCCTCACGCTACAGGCCCCTTGAGAAGTACGGGCTCAACGTTATG2832                           AlaProSerArgTyrArgProLeuGluLysTyrGlyLeuAsnValMet                               930935940                                                                      GGGCTCAAGCTCCTTCTTCGACGAACTCCGGAAGCTCGCCCGGAAGGA2880                           GlyLeuLysLeuLeuLeuArgArgThrProGluAlaArgProGluGly                               945950955960                                                                   AGCCGCCAGATGGGAGGTGCCCCCGGTGGGATCTTCTCCAGAACGAGA2928                           SerArgGlnMetGlyGlyAlaProGlyGlyIlePheSerArgThrArg                               965970975                                                                      ATGAACGCTATCCTCGGGAAGAGCCAGCGGAGGCTTCTCGCCCTAAAG2976                           MetAsnAlaIleLeuGlyLysSerGlnArgArgLeuLeuAlaLeuLys                               980985990                                                                      CCCCTCTCCTCGAACTCCCCCCTAAGCTCCTCCGGTGAGGGAAAGGCG3024                           ProLeuSerSerAsnSerProLeuSerSerSerGlyGluGlyLysAla                               99510001005                                                                    TCTATCGAGCTGACAAGGTGTTCGGCTTTCTCGTTGCCCGTCGTCAGC3072                           SerIleGluLeuThrArgCysSerAlaPheSerLeuProValValSer                               101010151020                                                                   TTTCCGATGAGGGGAACAACCGTTCTCGTGAGCCAGGCCATCTTTCCA3120                           PheProMetArgGlyThrThrValLeuValSerGlnAlaIlePhePro                               1025103010351040                                                               AGGAGCGAGGGGTTCTTTGAGAACTCGAGGATTACGAGCCTTCCTCCC3168                           ArgSerGluGlyPhePheGluAsnSerArgIleThrSerLeuProPro                               104510501055                                                                   GGCTTCAGAACACGGTGGAGCTCCTCTATAGCTTTTTCCCTATCGGAG3216                           GlyPheArgThrArgTrpSerSerSerIleAlaPheSerLeuSerGlu                               106010651070                                                                   AAGNTTCTGAGGTCGGAGGCGACGCTGACAATGTCGNAGCTCCCGTCC3264                           LysXaaLeuArgSerGluAlaThrLeuThrMetSerXaaLeuProSer                               107510801085                                                                   GGAGNCATTTCTTCCGCCCTGCCAACGCTCAGCCTCGCGNAGGGGACC3312                           GlyXaaIleSerSerAlaLeuProThrLeuSerLeuAlaXaaGlyThr                               109010951100                                                                   TTTCTCCCCGCTATCCTGAGCATCTCCTCGCTGCAGTCGAGGCCGAAC3360                           PheLeuProAlaIleLeuSerIleSerSerLeuGlnSerArgProAsn                               1105111011151120                                                               TACGCCCGACAGGTTTCTCTTTTCAAGCCTCTTCCTCATGCAGAGCAT3408                           TyrAlaArgGlnValSerLeuPheLysProLeuProHisAlaGluHis                               112511301135                                                                   CATGTCCCTGGTTCCGCAGGCCACGTCAAGGATTTTCGGCCTTTCGCG3456                           HisValProGlySerAlaGlyHisValLysAspPheArgProPheAla                               114011451150                                                                   AACCTCAAGGGACTTCAAAACCTCCTCGCAGGCCTTTTTCCTCCACAA3504                           AsnLeuLysGlyLeuGlnAsnLeuLeuAlaGlyLeuPheProProGln                               115511601165                                                                   CCTGTCGAGACTGAGGCTTATCAGCCTGTTGGTATCGTAGCGCTCCGC3552                           ProValGluThrGluAlaTyrGlnProValGlyIleValAlaLeuArg                               117011751180                                                                   AATGCTGTCAAAGAGCTCCCTTACCAAGCTCCTCCCTCCCGAGGACCT3600                           AsnAlaValLysGluLeuProTyrGlnAlaProProSerArgGlyPro                               1185119011951200                                                               TCTTTATCTTCGCGGGCCTTCCGCCTAGGTAAACCCTGTCCGCAATTT3648                           SerLeuSerSerArgAlaPheArgLeuGlyLysProCysProGlnPhe                               120512101215                                                                   AGAGCTCCTCGAGCTGGTGCGAGACGACCAGAACGCCTTTTCCAGAAT3696                           ArgAlaProArgAlaGlyAlaArgArgProGluArgLeuPheGlnAsn                               122012251230                                                                   TGGCTAATTCCCGAATTATGGAAAGAAGTTCTTCTTTGGAGCGCGCGT3744                           TrpLeuIleProGluLeuTrpLysGluValLeuLeuTrpSerAlaArg                               123512401245                                                                   CGAGGCCGGAGGGCTCGTCCAGAATCAGGACATCGAAGTCCAGCAGGG3792                           ArgGlyArgArgAlaArgProGluSerGlyHisArgSerProAlaGly                               125012551260                                                                   CGCGCAGGAGAGAAGTTTTTCTCCTCGTCCCCCCGCTCACTTCCTTTG3840                           ArgAlaGlyGluLysPhePheSerSerSerProArgSerLeuProLeu                               1265127012751280                                                               GATAGAGGTCGAGGTAGTTCTCCAGGCCGAGTCTCTCCACGTATTCGA3888                           AspArgGlyArgGlySerSerProGlyArgValSerProArgIleArg                               128512901295                                                                   GCCTGCACTCCCTTCCCCAGCGGGCCGGCAGGCAGACGTTGTCCCGCC3936                           AlaCysThrProPheProSerGlyProAlaGlyArgArgCysProAla                               130013051310                                                                   TCTTCCACGGCAGAAGGTAGTCCTCCTGATAGAGGACGGAGGGGTTCT3984                           SerSerThrAlaGluGlySerProProAspArgGlyArgArgGlySer                               131513201325                                                                   TCACAAAGACCTCGCCCGAGTCCGGGTTCTCAACTCCGGCCAAAATCT4032                           SerGlnArgProArgProSerProGlySerGlnLeuArgProLysSer                               133013351340                                                                   TCACGAGGGTGCTCTTTCCCGTCCCGTTCGGCCCGAGGCCGACCACCT4080                           SerArgGlyCysSerPheProSerArgSerAlaArgGlyArgProPro                               1345135013551360                                                               CGCCGGCCCCCTCAAGGCCCCCGTCGAGTATGGGCTCACAGGACTTTC4128                           ArgArgProProGlnGlyProArgArgValTrpAlaHisArgThrPhe                               136513701375                                                                   GGTTCTTCACCAGTACCAGCCTTTCACCCATTCCTCGACCTCCTCAGA4176                           GlySerSerProValProAlaPheHisProPheLeuAspLeuLeuArg                               138013851390                                                                   AGTAGCTGGTCGAGGGTTATCATGAGCAGGATTAAGAGCAGTGCCCAG4224                           SerSerTrpSerArgValIleMetSerArgIleLysSerSerAlaGln                               139514001405                                                                   GCAAAAACACCGGCTTTAATTCCCAAATCGGAGACGAGCTGGCCGATT4272                           AlaLysThrProAlaLeuIleProLysSerGluThrSerTrpProIle                               141014151420                                                                   CCTCCCGCTGAACCAAAAGCCTCGGCAACGACGCTTATCCTGAGCGCT4320                           ProProAlaGluProLysAlaSerAlaThrThrLeuIleLeuSerAla                               1425143014351440                                                               ATTCCCAGGGCGACTCTGCCTGCCGAGACCATCTCGGGGAGCGTTCCC4368                           IleProArgAlaThrLeuProAlaGluThrIleSerGlySerValPro                               144514501455                                                                   GGGACGATGAAGTGCCGGAGCAGCTTTGAGGGTTTGAGGAGAACTATC4416                           GlyThrMetLysCysArgSerSerPheGluGlyLeuArgArgThrIle                               146014651470                                                                   AGCGGGCGGTATTTTTCTATCACCTTTTCGCTTGAGCTCACTCCAGC4463                            SerGlyArgTyrPheSerIleThrPheSerLeuGluLeuThrPro                                  147514801485                                                                   (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1487 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (v) FRAGMENT TYPE: internal                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        AsnSerArgMetAsnLeuIleTrpSerValLeuSerLeuTyrIlePro                               151015                                                                         GluProMetGlyPheSerLeuLeuAlaTyrPheIleGlySerLeuMet                               202530                                                                         SerPheGlnMetCysSerGlyThrIleGlyIleTrpSerGlnSerHis                               354045                                                                         SerAlaCysGlySerLeuLysMetLysSerThrValLeuSerSerThr                               505560                                                                         ThrLeuThrSerLeuSerGlnProGlnThrLeuGluLysGluAlaPro                               65707580                                                                       ValTrpPhePheAlaArgLysValAsnLeuThrSerSerAlaValArg                               859095                                                                         GlyPheProSerCysGlnValArgProSerPheSerLeuThrSerVal                               100105110                                                                      LeuSerLeuIleHisPheProAspSerAlaSerGlnGlyMetSerTrp                               115120125                                                                      SerPheSerGlySerLysAsnArgGlySerMetArgLeuSerCysLeu                               130135140                                                                      LeuArgProArgArgGlnGlyAspSerSerSerAlaAspSerThrAsp                               145150155160                                                                   ArgLeuGlnArgArgGlyPheHisSerTrpGluAlaProArgArgGly                               165170175                                                                      ArgSerProArgIleGlnGlnGlyLeuLysProSerAlaLeuProAla                               180185190                                                                      AlaAsnGlnGlyPheValSerCysAlaProProGlnThrAlaArgVal                               195200205                                                                      CysSerTyrLysAsnAlaLeuGlnIleTyrLysThrLeuGlySerVal                               210215220                                                                      IleLysLeuGlyGlnLysLeuLysSerAlaAsnLeuIleArgGluLys                               225230235240                                                                   GlyLysValThrTrpGlyLeuValTyrAlaThrArgArgLeuSerLeu                               245250255                                                                      GlyArgValProValArgLeuSerValArgAspGlyArgGlnAlaGln                               260265270                                                                      GluGluHisSerGluHisArgLeuValGluValGlyGlnGlySerLeu                               275280285                                                                      GlnHisLysGluGlyThrArgGlnArgArgProAlaArgGlyGlyAsp                               290295300                                                                      LysGlnLeuArgThrLeuArgGluGlySerProProArgGlnArgPro                               305310315320                                                                   ArgSerGluArgLeuGlnAspTrpAsnArgValGluGlnAspLeuSer                               325330335                                                                      LeuAlaAsnValValCysGlyGlyArgAlaGlyGlnLeuArgThrArg                               340345350                                                                      GluGlyArgGlnAsnArgArgHisAlaArgArgAlaArgArgAspSer                               355360365                                                                      GluSerSerGlyAspSerLeuLeuProProArgTyrArgAlaProGln                               370375380                                                                      GlyAlaGlyLeuGlnGlyHisArgGluProGlnProLeuHisAlaPro                               385390395400                                                                   ProLeuAlaSerArgSerAspAsnArgGluGlyGluGlyProHisGln                               405410415                                                                      ArgAspTrpLeuGlyArgAlaGlyGluArgGlyGlyValArgGlnVal                               420425430                                                                      ArgGlyValHisArgGluArgThrArgGlyProArgTyrValGluHis                               435440445                                                                      LeuGlnArgAlaAspGlyArgCysGlyAlaArgLeuProArgAlaLeu                               450455460                                                                      LeuArgLeuSerAlaGlyGlyTyrGluProArgGlyGlyLysAlaGly                               465470475480                                                                   AsnProGlnHisAspLysArgProArgThrGlyLeuGlnAspAspLys                               485490495                                                                      GluValArgGlnGlyLysGlyArgGlyPheProLeuArgGlyArgGly                               500505510                                                                      ArgAspAsnLeuGlnGlnHisArgArgCysLeuSerIleArgLeuGln                               515520525                                                                      ArgProLysGlyArgGluSerCysArgLysArgGlnLeuLeuProGln                               530535540                                                                      ArgAlaLeuLeuArgArgAsnProGlnGlyGlnAlaGlnHisArgVal                               545550555560                                                                   ArgArgAspLeuArgGlnSerSerAlaSerGlnGlyGluArgLeuAsp                               565570575                                                                      ArgArgLeuLeuHisGluArgSerArgGlnValPheGlyAlaGlnVal                               580585590                                                                      ProGluHisThrProAspIleLeuProGlySerSerGlnLeuArgLeu                               595600605                                                                      ArgLeuGlnAlaArgGluPhePheArgArgArgLysAlaArgLysArg                               610615620                                                                      HisArgLeuGlyAspLeuSerGlyGlyAspLeuArgLeuAspLysArg                               625630635640                                                                   GlyGlnGlnIleArgGlyProGlyLeuArgHisArgLysArgAsnSer                               645650655                                                                      ArgPheAsnHisProAlaAlaValLeuProArgGluProCysSerGlu                               660665670                                                                      AspGlyGlyValArgGlyGlyLeuArgArgGlnGlyLeuProLeuLeu                               675680685                                                                      GlyAlaAspArgGlnLeuArgValGlyProArgPheGlnAspGluVal                               690695700                                                                      ArgProLeuSerGlySerHisAsnGlnGlyGluAsnThrAlaGlyGly                               705710715720                                                                   LysArgLysGlyLeuGlyHisArgGlyGluGlnArgSerGluGlnGly                               725730735                                                                      AsnProGlyGluValArgThrTrpValLysValMetLysThrIleAla                               740745750                                                                      ValAspGluAspThrTrpGluAlaArgSerArgSerGlyLeuArgGln                               755760765                                                                      IleValArgArgSerProGluLysAlaHisThrGlyLeuAspArgVal                               770775780                                                                      AspSerThrArgProArgAlaAlaThrThrArgArgProSerSerCys                               785790795800                                                                   SerThrSerArgThrArgArgArgGluAspArgValMetLysArgLeu                               805810815                                                                      ProGluArgValSerPheAspProGluAlaPheValGluIleAsnArg                               820825830                                                                      LysArgAsnArgAspPheLeuGluPheLeuLeuAlaGluPheGlnVal                               835840845                                                                      XaaValSerPhePheThrValHisProTyrLeuLeuGlyLysThrTyr                               850855860                                                                      LeuGlyArgAspLeuGluSerGluValArgAlaLeuAsnGluAlaTyr                               865870875880                                                                   ThrIleValTyrProThrLysGluLeuLeuMetArgAlaIleGluIle                               885890895                                                                      GluAlaArgLeuIleLysArgGlyIlePheLeuSerPheAspAspIle                               900905910                                                                      ValIleGlyValThrAlaIleGluAsnAsnAlaLeuLeuValSerSer                               915920925                                                                      AlaProSerArgTyrArgProLeuGluLysTyrGlyLeuAsnValMet                               930935940                                                                      GlyLeuLysLeuLeuLeuArgArgThrProGluAlaArgProGluGly                               945950955960                                                                   SerArgGlnMetGlyGlyAlaProGlyGlyIlePheSerArgThrArg                               965970975                                                                      MetAsnAlaIleLeuGlyLysSerGlnArgArgLeuLeuAlaLeuLys                               980985990                                                                      ProLeuSerSerAsnSerProLeuSerSerSerGlyGluGlyLysAla                               99510001005                                                                    SerIleGluLeuThrArgCysSerAlaPheSerLeuProValValSer                               101010151020                                                                   PheProMetArgGlyThrThrValLeuValSerGlnAlaIlePhePro                               1025103010351040                                                               ArgSerGluGlyPhePheGluAsnSerArgIleThrSerLeuProPro                               104510501055                                                                   GlyPheArgThrArgTrpSerSerSerIleAlaPheSerLeuSerGlu                               106010651070                                                                   LysXaaLeuArgSerGluAlaThrLeuThrMetSerXaaLeuProSer                               107510801085                                                                   GlyXaaIleSerSerAlaLeuProThrLeuSerLeuAlaXaaGlyThr                               109010951100                                                                   PheLeuProAlaIleLeuSerIleSerSerLeuGlnSerArgProAsn                               1105111011151120                                                               TyrAlaArgGlnValSerLeuPheLysProLeuProHisAlaGluHis                               112511301135                                                                   HisValProGlySerAlaGlyHisValLysAspPheArgProPheAla                               114011451150                                                                   AsnLeuLysGlyLeuGlnAsnLeuLeuAlaGlyLeuPheProProGln                               115511601165                                                                   ProValGluThrGluAlaTyrGlnProValGlyIleValAlaLeuArg                               117011751180                                                                   AsnAlaValLysGluLeuProTyrGlnAlaProProSerArgGlyPro                               1185119011951200                                                               SerLeuSerSerArgAlaPheArgLeuGlyLysProCysProGlnPhe                               120512101215                                                                   ArgAlaProArgAlaGlyAlaArgArgProGluArgLeuPheGlnAsn                               122012251230                                                                   TrpLeuIleProGluLeuTrpLysGluValLeuLeuTrpSerAlaArg                               123512401245                                                                   ArgGlyArgArgAlaArgProGluSerGlyHisArgSerProAlaGly                               125012551260                                                                   ArgAlaGlyGluLysPhePheSerSerSerProArgSerLeuProLeu                               1265127012751280                                                               AspArgGlyArgGlySerSerProGlyArgValSerProArgIleArg                               128512901295                                                                   AlaCysThrProPheProSerGlyProAlaGlyArgArgCysProAla                               130013051310                                                                   SerSerThrAlaGluGlySerProProAspArgGlyArgArgGlySer                               131513201325                                                                   SerGlnArgProArgProSerProGlySerGlnLeuArgProLysSer                               133013351340                                                                   SerArgGlyCysSerPheProSerArgSerAlaArgGlyArgProPro                               1345135013551360                                                               ArgArgProProGlnGlyProArgArgValTrpAlaHisArgThrPhe                               136513701375                                                                   GlySerSerProValProAlaPheHisProPheLeuAspLeuLeuArg                               138013851390                                                                   SerSerTrpSerArgValIleMetSerArgIleLysSerSerAlaGln                               139514001405                                                                   AlaLysThrProAlaLeuIleProLysSerGluThrSerTrpProIle                               141014151420                                                                   ProProAlaGluProLysAlaSerAlaThrThrLeuIleLeuSerAla                               1425143014351440                                                               IleProArgAlaThrLeuProAlaGluThrIleSerGlySerValPro                               144514501455                                                                   GlyThrMetLysCysArgSerSerPheGluGlyLeuArgArgThrIle                               146014651470                                                                   SerGlyArgTyrPheSerIleThrPheSerLeuGluLeuThrPro                                  147514801485               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What is claimed is:
 1. A method for providing a thermostable enzyme having improved enzyme activities as compared to a corresponding wild-type enzyme at lower temperatures, comprising:(a) subjecting at least one polynucleotide encoding an enzyme which is stable at a temperature of at least 60° C. to a random mutagenesis; and (b) screening mutant enzymes produced in (a) for a mutated enzyme or for a polynucleotide encoding a mutated enzyme, wherein the mutated enzyme is stable at a temperature of at least 60° C.; has an enzyme activity at a temperature at least 10° C. below its optimal temperature range; and has activity greater than the enzyme encoded by the polynucleotide of step (a) prior to mutagenesis.
 2. The method of claim 1, wherein the enzyme is selected from the group consisting of lipase, esterase, protease, glycosidase, glycosyl transferase, phosphatase, kinase, monoxygenase, dioxygenase, haloperoxidase, lignin peroxidase, diarylpropane peroxidase, epoxide hydrolase, nitrite hydratase, nitrile nitrilase, transaminase, amidase and acylase. 